外周血造血干细胞不同保存方法的比较

目的为造血干细胞低温保存选择合适的方法.方法20例外周血单个核细胞加入DMSP或DMSO加HES,-80℃冰箱(简称冰箱)或程控降温,冰箱或液氮保存,定期检测标本的CFU-GM,LTC-CD34 ,TBR,计回收率.结果冰箱降温并保存,5%DMSO-6%HES为保护剂的CFU-GM、LTC-IC、CD34 、TBR回收率分别为82.2%±14.7%、83.0%±12.2%、94.2%±4.3%、97.7%±3.9%,比10%DMSO者具体回收率明显高(P＜0.05);同一种保护剂下冰箱降温并保存与程序降温冰箱保存比,差异不显著(P＞0.05),冰箱降温并保存、冰箱降温液氮保存及程控降温液氮保存,在一年内,各种回收率差异不显著(P＞0.05),二年时,只有冰箱降温并保存者其各种回收率指标明显下降(P＜0.05);细胞复温后,标本不稀释也不洗涤,室温下放置,细胞活力的各指标均下降(P＜0.05),以含10%DMSO为保护剂者影响最大.结论造血干细胞优选保存方法是保护剂用5%DMSO-6%HES,保存时间不超过一年时,用冰箱降温并保存,长期保存时,冰箱降温则需液氮保存,细胞复温后,标本内保护剂应立即稀释(临床应用)或去除.     

The fragile X mutation does not have any major effect on the expression of the hypoxanthine phosphoribosyltransferase (HPRT) locus in human fibroblasts

Summary The possible influence of the fragile X mutation at Xq27 on the expression of the neighbouring gene (at Xq26) for hypoxanthine phosphoribosyl transferase (HPRT) was studied by determination of the levels of HPRT-RNA and HPRT enzyme activity in fibroblast cell cultures from 7 fragile X patients. These levels were lower (although not statistically significantly lower) than in normal fibroblast cultures. Hence, these data do not support the notion of a major effect of the fragile X mutation on the expression of the HPRT gene.     

Effects of peripheral blood stem cell apheresis on systemic cytokine levels in patients with multiple myeloma

BACKGROUND AIMS. Pro-angiogenic cytokines can affect myeloma cell proliferation directly and indirectly through stimulation of cancer-associated angiogenesis. METHODS. We investigated how peripheral blood stem cell (PBSC) collection affected plasma angioregulatory cytokine levels in 15 consecutive myeloma patients. RESULTS. Plasma levels of hepatocyte growth factor (HGF) were significantly increased prior to apheresis in patients compared with donors, and a further increase was detected immediately after PBSC apheresis. HGF levels decreased within 24 h, but were still higher than the levels in healthy donors, whose HGF levels were not altered by platelet apheresis. Pre-apheresis levels of other angioregulatory cytokines, angiopoietin-2 and vascular endothelial growth factor (VEGF), were also increased in patients, whereas angiopoietin-1, angiogenin and basic fibroblast growth factor levels did not differ from healthy controls. PBSC harvesting decreased angiopoietin-1 and VEGF levels, increased the microvascular endothelial cell marker endocan levels but did not affect the other mediators. CONCLUSIONS. Our results show that PBSC apheresis alters systemic angioregulatory profiles in myeloma patients. This cytokine modulation is not a general characteristic of all apheresis procedures and was not seen in healthy platelet donors.     

Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy

Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50?%. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.     

Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73

Purinergic Signalling - CD39 and CD73 are ecto-nucleotidases present on human peripheral blood mononuclear cells (PBMCs) and are emerging biomarkers on these cells in various disorders including...     

The use of CD 34(+) mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates

In utero hematopoietic stem cell transplantation is a therapeutic procedure that could potentially cure many developmental diseases affecting the immune and hematopoietic systems. In most clinical and experimental settings of fetal hematopoietic transplantation the level of donor cell engraftment has been low, suggesting that even in the fetus there are significant barriers to donor cell engraftment. In postnatal hematopoietic transplantation donor cells obtained from mobilized peripheral blood engraft more rapidly than cells derived from marrow. We tested the hypothesis that use of donor hematopoietic/stem cells obtained from mobilized peripheral blood would improve engraftment and the level of chimerism after in utero transplantation in non-human primates. Despite the potential competitive advantage from the use of CD 34(+) from mobilized peripheral blood, the level of chimerism was not appreciably different from a group of animals receiving marrow-derived CD 34(+) donor cells. Based on these results, it is unlikely that this single change in cell source will influence the clinical outcome of fetal hematopoietic transplantation.     

The distribution of the major peripheral blood T, B and NK cell subsets does not predict the clinical outcome of Graves' disease patients after methimazole therapy

Biological markers capable of predicting the clinical outcome of antithyroid drug therapy could be clinically useful in selecting the modality of treatment for Graves' disease, but at present they are unavailable. In the present study we prospectively explore the value of 22 different peripheral blood T, B and NK lymphocyte subsets to predict remission and relapse in a group of 42 Graves' disease patients. Eighteen patients were studied at diagnosis, before treatment, and 24 during antithyroid drug therapy. All cases were followed-up for at least one year after finishing an 18 month cycle of methimazole therapy. The combination of flow cytometry and 3- color immunofluorescence did not reveal significant differences in the distribution of the major peripheral blood T, B and NK cell subsets between the relapsed patients and those in remission, both in the groups studied at diagnosis and in those analyzed during the cycle of antithyroid drug therapy. In our search for a prognostic marker for relapse prediction we found that some lymphoid subpopulations such as total B cells, total NK and NK CD8+ cells showed high sensitivity (88-100%). In turn, other subsets such as TCD8+, total T and B cells expressing the CD25 antigen displayed high specificity (77-88%).     

Beneficial effects of fetal-maternal microchimerism on the activated haplo-identical peripheral blood stem cell treatment for cancer

Background Previous studies have shown that activated haplo-identical peripheral blood stem cell (haplo-PBSC) treatment exerts an anti-tumor effect on patients with metastatic solid tumors. The purpose of this study was to test the hypothesis that fetal-maternal microchimerism enhances the beneficial effect of the haplo-PBSC treatment for cancer. Methods Twenty-five patients with advanced-stage solid tumors refractory to standard chemotherapy were treated with haplo-PBSC donated by parents or children. Fetal-maternal microchimerism status was determined using nested polymerase chain reaction typing using sequence-specific primers (PCR-SSP). Clinical outcomes, including therapeutic response by measuring tumor size changes using CT scanning and survival times, were evaluated. The donor-recipient mixed lymphocyte response (MLR) was detected using an MTT proliferation assay. Cytokine production was determined using an ELISA method. Results Six patients receiving maternal-child transplants were fetal-maternal microchimerism positive (+). The mean survival time of patients with the microchimerism(+) haplo-PBSC treatment was 30.17+/-5.32 months (median 17 months), which was significantly longer than that of patients with negative (-) microchimerism (mean 16.95+/-3.29 months, median 8 months; P=0.043). The therapeutic response rate was significantly higher in microchimerism(+) patients (83.3%) than that in microchimerism(-) patients (36.8%) (P=0.047). Furthermore, suppression of donor-recipient MLR and increased production of a T-helper type 1 (Th1) type cytokine, interferon (IFN)-gamma, were found in microchimerism(+) patients after haplo-PBSC treatment. Discussion This small study suggests that fetal-maternal microchimerism is associated with a statistically significant improvement in anti-tumor effect of activated haplo-PBSC treatment. Further study is required to elucidate the mechanism for this observation.     

Comparative analysis of different flow cytometry-based immunophenotypic methods for the analysis of CD59 and CD55 expression on major peripheral blood cell subsets

BACKGROUND: Flow cytometry-based immunophenotypic techniques for the analysis of CD55 and CD59 expression on the major cell populations present in blood are the preferred method for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH). METHODS: In the present study, we comparatively analyze the effects of stain-lyse-and-then-wash techniques and lyse-wash-and-then-stain procedures on the detection of both CD55 and CD59 expression on the major peripheral blood (PB) leucocyte subsets, as analyzed by flow cytometry. Our major goal was to establish the minimum amounts of anti-CD55 and anti-CD59 reagents required to be added to a minimum volume of blood, which would allow an optimal staining for both antigens on red cells, platelets, and leucocytes present in a single tube. RESULTS: Our results show that upon comparing stain-lyse-and-then-wash techniques with lyse-wash-and-then-stain protocols, the presence of important amounts of red cells at the time peripheral blood leucocytes are stained for CD55 and CD59 is associated with a significantly (P < 0.01) lower and more heterogeneous pattern of antigen expression on almost all major PB leucocyte subsets, supporting the need to use red cell lysing procedures prior to the staining of leucocytes. Identical, optimal patterns of antigen staining for CD55 and CD59 were obtained upon incubating 3 microL of blood with 10 microL of each of these monoclonal antibody (mAb) reagents (protein concentration of 0.05 microg/microL and 0.2 microg/microL respectively) for 30 min (room temperature [RT]) using a non-lyse-non-wash sample preparation procedure. This latter procedure allowed for the simultaneous analysis of CD55 and CD59 expression on red cells, platelets, neutrophils, monocytes, and lymphocytes present in the sample through the combined staining of CD55 and CD59 with CD64-fluorescein isothiocyante (FITC) plus CD61-peridinin chlorophyll protein (PerCP) and CD45-PerCP. CONCLUSIONS: In summary, our results show that the sample preparation protocol has a significant impact on the quality of the staining obtained for the CD55 and CD59 antigens on the major PB leucocyte subsets; additionally, we propose a simple and reliable stain-non-lyse-non-wash method for the simultaneous analysis of CD55 and CD59 expression on PB red cells, platelets, neutrophils, monocytes, and lymphocytes, which could be reached through the use of two triple stainings.     

自体外周血干细胞移植联合髓芯减压治疗早中期股骨头缺血性坏死的临床研究

目的对比研究单纯股骨头髓芯减压与联合自体外周血干细胞移植治疗早中期股骨头缺血性坏死（ANFH）的临床疗效。方法 2004年10月至2012年3月期间就诊于福建省龙岩市第一医院,经影像学检查及术后病理检查确诊为ANFH的患者90例。A组采用单纯髓芯减压共34例,男22例,女12例,年龄22~62岁（中位年龄41.5岁）。B组采用髓芯减压联合自体外周血干细胞移植（用rhG-CSF皮下注射4 d进行外周血干细胞动员,第5天分离外周造血干细胞混悬液。在髓芯减压基础上沿减压隧道注入自体外周血干细胞10~15 ml）56例,男35例,女21例,年龄21~62岁（中位年龄39岁）。按世界骨髓循环研究学会（ARCO）国际骨坏死标准：A组：Ⅰ期7髋,Ⅱ期26髋,Ⅲ期17髋;B组：Ⅰ期10髋,Ⅱ期50髋,Ⅲ期29髋。术后及随访期间进行Harris评分和疼痛视觉模拟（VAS）评分,对X线片、CT及MRI检查结果进行评估。两组Harris评分和VAS评分比较用成组t检验。结果 A组34例,B组49例随访13~89个月,平均38.5个月。术后3、6、12个月与术前的Harris评分比较有明显提高,VAS评分有明显的下降;术前、术后3个月两时间点的A、B组Harris评分比较差异无统计学意义（t=0.342、0.628,P=0.781、0.516）;术后6、12个月的B组评分优于A组（t=2.991、7.753,P=0.009、0.001）。术后12个月A、B两组T1相低信号区所占股骨头体积的比例分别为（17.24±3.71）﹪和（12.11±3.14）﹪,差异有统计学意义（t=4.360,P=0.001）。结论髓芯减压联合自体外周血干细胞移植治疗早中期ANFH,可显著减轻关节疼痛,改善股骨头血液供应,明显恢复关节功能,加速病灶区骨组织修复,有效防止股骨头进一步塌陷。     

Upregulation of TLR2 expression on G-CSF-mobilized peripheral blood stem cells is responsible for their rapid engraftment after allogeneic hematopoietic stem cell transplantation

Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSCs) are more frequently used as the cellular source in allogeneic hematopoietic stem cell transplantation (HSCT) than bone marrow stem cells (BMSCs) because they promote more rapid engraftment and immune reconstitution. However, the underlying mechanism for this is not fully understood. Here, we investigated the role of Toll-like receptor 2 (TLR2) on PBSCs in promoting rapid engraftment after allogeneic HSCT. We found that PBSCs highly expressed TLR2 in comparison to BMSCs, and TLR2 was directly induced by G-CSF signaling. Treatment with the TLR2 ligand, Pam(3)CSK(4) (PAM), more efficiently induced myeloid differentiation of PBSCs than BMSCs. Similarly, endogenous TLR2 ligands from the serum of recipients of allogeneic transplantation more rapidly stimulated myeloid differentiation of PBSCs compared with BMSCs. PAM treatment of TLR2(-/-) syngeneic recipient mice transplanted with PBSCs resulted in significantly elevated numbers of PBSC-derived myeloid cells and spleen colony formation compared with controls. Our results demonstrate that TLR2 signaling in PBSCs correlates with their ability to rapidly differentiate into myeloid cells, resulting in improved engraftment. Thus, TLR2 may be a novel target for increasing the efficiency of allogeneic HSCT by overcoming engraftment failure or delayed engraftment.     

Isw1a does not have strict limitations on the length of extranucleosomal DNAs for mobilization of nucleosomes assembled with HeLa cell histones

The Saccharomyces cerevisiae Isw1a and Isw2 ATP-dependent chromatin-remodeling complexes have important roles in vivo in the regulation of nucleosome positioning and modulation of gene activity. We studied the ability of the Isw1a- and Isw2-remodeling enzymes to reposition nucleosomes in mono- and dinucleosomes templates with variably positioned histone octamers (in the center or at the ends of the DNA fragment). To compare the Isw1a and Isw2 nucleosome-mobilizing activities, we utilized mono- and dinucleosome templates reconstituted with purified HeLa cell histones and DNA containing one or two copies of the “601” nucleosome high-affinity sequence used to specifically position nucleosomes on the DNA. The obtained data suggest that Isw1a is able to mobilize HeLa cell histone-assembled mononucleosomes with long (more than 30?bp) extranucleosomal DNAs protruding from both sides, which contrasts to the previously reported inability of Isw1 to mobilize similar nucleosomes assembled with recombinant yeast histones. The results also suggest that Isw1a and Isw2 can mobilize nucleosomes with unfavorably short linker DNA lengths, and the presence of internucleosomal interactions promotes mobilization of nucleosomes even when the linkers are short.